1. Field of the Invention
The invention generally relates to the field of microscopy and, more specifically, to Resonant Stimulated Raman Scattering Microscope.
A new non-linear optical (NLO) effect is disclosed, namely a Resonant Stimulated Raman Scattering (RSRS) effect for a new microscope arising from the excess vibrations generated from Resonant Raman (RR) processes in native (intrinsic) or extrinsic absorber, for example β-carotene or Flavins, transfer to its host media (say methanol) or tissues to excite the associated vibrations of the solutions or tissues via anharmonic vibrational interactions. This process leads to Raman gain in resonance to energy transfer from absorber and host vibrations, which are close to absorber, to host media solution or tissue. The Raman gain is enhanced from resonance of Raman cross section when pump photon frequency ωL is in absorption or in wing near the emission wavelength of the intrinsic or extrinsic molecules to enhance the signal gain of the Stokes photon ωs beam. The RSRS effect is important because it greatly increases the signal in stimulated Raman microscopy using RR of Flavins in the brain, breast, Cervix, skin, other organs and arteries. Using two beams, one at resonance of the molecules say at 532 nm or other visible pump laser light (i.e. 524 nm, 488 nm) and the other Stokes light beam (a tunable laser beam) at well defined vibrational shift of CH2, CH3 and amide 1 and tryptophan (amino acids) modes to get lipids and proteins vibrational lines in an image of the media—tissue, cell, or solution—for enhanced Stimulated Raman signal Gain at Stokes or Loss at pump laser in the microscope. Using objective lens with high NA, the beams are scanned by a scanner across the sample surface (x,y) and moved in depth z to get 2D or 3D plots of vibrational maps. The laser pump and tunable Stoke beam differ by the vibrational frequency ωq to image, for example to detect and image biomolecules of glucose, tryptophan, amino acids, lipids, proteins, analytes, cholesterol in tissue, cells and bio fluids (urine and blood).
Resonant Raman Scattering (RSRS), combining both Resonant Raman Scattering (RRS) and Stimulated Raman Scattering (SRS) processes, generates a first new non-linear optical (NLO) effect.
The RSRS scattering process is at the heart of the new microscope for imaging and detecting changes associated with vibrations with disease. The observation of RSRS presented here is most important for new Stimulated Raman Loss (SRL) and Stimulated Raman Gain (SRG) microscopes in order to enhance signals of images from vibrations in biomedical tissues, cells fluids, and chemicals in the samples (ex vivo and in vivo). The selection of the pump or Stokes near an electronic resonance will improve the signal and the signal to noise ratio (i.e., S/N) of the SRS microscope image for tissues and cells from brain, breast, cervix, skin, arteries and in urine spinal, eye fluids and blood, etc.
2. Description of the Prior Art
Raman scattering is one of the key optical spectroscopic processes arising from inelastic scattering of light with vibrations in materials. The scattered light has a characteristic frequency shill due to vibrations accompanied by generation of optical phonons in the material. The Raman effect has been an active topic in various fields of science since its discovery in 1928 by Raman and Krishnan. The advent of laser in the 60's the Raman effect exploded in use. The Raman process occurs when a photon is scattered from a vibrational mode having its energy difference from the incident beam by the vibrational frequencies.
There are several different types of Raman processes that can occur, depending on the types of interactions between laser and matter, such as spontaneous, resonance, hyper Raman, and stimulated Raman. Spontaneous Raman (sR), despite being the weakest form of scattering, has widely been used as a powerful technique to investigate complex molecular and solid-state systems [2,3]. An enhancement of the Raman signal, essential for studies at low concentrations or in low cross section compounds, is achieved by Resonant Raman Spectroscopy (RRS), in which the laser excitation wavelength is tuned to match the energy of any electronic transitions of a system. Stimulated Raman scattering (SRS) can occur when Stokes photons are generated by gain of sR scattering in forward and backward with high pump lasers. SRS was first discovered when a cell with nitrobenzene was introduced inside a ruby laser cavity [4], where a rather strong emission at the wavelength other than the fundamental wavelength (694.3 nm) was observed. Stoicheff's group [5] measured various regions in Raman processes at different laser pump intensities of the first Stokes in nitrogen and oxygen liquids, namely R, small SRS gain, SRS, and SRS saturation [5], as the pump laser intensity was increased. Several researchers have demonstrated different Raman gains from transient to transient depending on the pulse duration and vibrational lifetime under picosecond (ps) pulses [6]. In the early 1970's, the 4 wave interactions producing the white light continuum and competing with SRS spanning the visible and part of NIR, now called supercontinuum (SC), was discovered by Alfano and Shapiro [7] in solids and liquids using ps-pulses.
Today, the use of SRS gain and loss (G/L) is active for imaging vibration of lipids, proteins and other molecules in biological and chemical materials such as brain, breast, biofluids, cells and cancer by injecting both light at wavelengths of the pump and Stokes wave together at the input [8-12].
In SRS microscopy, the sample is coherently driven by two lasers: one is the pump beam with frequency ωL and the other is the Stokes beam with frequency ωs, where the difference is equal to a particular Raman-active molecular vibration of the sample. The SRS signals, including both stimulated Raman loss (SRL) at the laser pump beam and stimulated Raman gain (SRG) at the Stokes beam, are generated due to the nonlinear interaction between the photons and the vibration of the molecules [5, 6] for imaging [8-12]. The RSRS microscopy used either the pump or Stokes beam frequency to be in the electronic absorption band of the material for vibration enhancement via Raman cross section from the denominator poles. The development of novel nonlinear vibrational spectroscopy has allowed broadband SRS to provide high intensity coherent signal with low fluorescence background. In SRS, the sample is interrogated by a pair of overlapped narrowband picosecond (ps) Raman pulses and/or broadband femtosecond (fs) probe pulses. In SRS G/L process the vibrational spectrum, for example from lipids and proteins, occurs with the incoherent fluorescence background and the electronic susceptibility is efficiently suppressed. There is a need to sort out ways to increase the signal to noise (S/N) ratio in Stimulated Raman microscope that has been overlooked. Using Resonant Stimulated Raman scattering microscope occurs when one of the beam Pump or Stokes photons is it resonance with molecule electronic states to enhance the SRS cross section effect. In addition S/N is improved by higher frequency modulation that reduces the 1/f noise and dark current. The Stokes beam for ps/fs sources are created from the pump laser beam by OPO, OPA, or SHG to reduce jitter time effects between the pump and Stokes beams.